Detection of deltamethrin resistance in Rhipicephalus microplus ticks infesting cattle by adult immersion test

Introduction

Ticks are responsible for the production losses of animals by causing tick worry, tick toxicity, sucking of blood, irritancies, damage to hides and weight loss (Furman and Loomis, 1984; Patel et al., 2013) [12,27] . Tick-borne diseases ranked in fourth position in major infections of livestock (Ghosh et al., 2007) [14] . Economic losses due to tick-transmitted diseases and expenses on control of ticks reduce the cost-effectiveness of animal production. (Singh et al., 2022). Amongst the different tick species Rhipicephalus microplus is the most prevalent tick species found in India (Patel et al. 2013; Singh and Rath, 2013) [27,35] . Infestation of cattle tick Rhipicephalus microplus (R. microplus) is the most predominant tick species in Nagpur district of Maharashtra. (Manmod and Jumade, 2025) [25] . Tick control is done by different methods of which, the use of chemicals is the most commonly used method. Chemical control of ticks comprises the use of various acaricides available in the market. Acaricides include pesticides that kill the members of order Acarina, which includes ticks and mites (Wyk et al., 2016) [41] . Various classes of acaricides used for tick control are natural and synthetic pyrethrins, organochlorides, organophosphate, carbamate, neonicotinoid, formamidine, semicarbazone, phenylpyrazole, and miscellaneous. These acaricides act systemically by direct contact with external applications, or by fumigants or repellents. As the chemical acaricides plays an efficient role in tick control, the whole world is facing the problem of environmental pollution and acaricidal resistance. Resistance to acaricide is a decreased susceptibility of a parasite to acaricide when it is used at the recommended concentration for which the parasite was previously susceptible. Long-term use of acaricides, improper dilution, wrong application techniques, continuous, indiscriminate use and overdosing have resulted in acaricide resistance development (Abbas et al., 2014; Ravindran et al., 2011) [1,28] . The development of acaricidal resistance made it difficult to manage and control ticks and tick-borne diseases. (Guerrero et al., 2014) [17] . Acaricidal resistance development seen worldwide as well as in India. Difficulties and expenses involved in developing new acaricide make this topic of research more important (Abbas et al., 2014; Vudriko et al., 2016; Yessinou et al., 2018, Vatsya, et al., 2011, Shyma et al., 2015, Gaur et al., 2016, Kumar et al., 2017) [1,40,42,38,33,13,24] . By taking all these points in consideration, this research was planned to assess deltamethrin resistance status in Rhipicephalus microplus ticks infesting cattle from Nagpur district of Maharashtra.

Materials and Methods

The chemical acaricide from the synthetic pyrethroid group namely; deltamethrin (commercially available) procured from the local market of the area of the study was used in different working concentrations by diluting it with distilled water to evaluate the acaricidal resistance status in R. microplus ticks.

Collection of ticks

The study of assessment of deltamethrin resistance in cattle ticks was conducted in five talukas of Nagpur district of Maharashtra state namely; Nagpur, Kamptee, Ramtek, Kalmeshwar and Umred. About ten animals infested with ticks from each of four villages of each district were selected for tick collection. Ticks were collected from different organized and unorganized farms in ventilated, escape-proof sample collection bottles from body of cattle, from the ground, cracks, crevices and under stones of cattle shade. The history of spraying of deltamethrin (yes/no), its frequency, spraying interval, dose, and effectiveness against ticks was asked to the animal owner during tick collection. The ticks were kept cool and transported to the Department of Veterinary Parasitology laboratory for further studies. During the summer season, sterilized sand was kept at the bottom of the bottles on which filter paper and a few drops of water were sprinkled to maintain the moisture level.

In vitro assessment of detection of deltamethrin resistance

The acaricidal activity of deltamethrin against adult ticks was evaluated following the adult immersion test (AIT) as per the protocol described by FAO, (2004) [10] and Drummond et al. (1973) [9] with slight modifications. The ticks were weighed and the ticks with approximately similar weights were grouped into six groups each containing six female ticks. The engorged female ticks were immersed in a 1% sodium hypochlorite solution for 1 min, rinsed with sterile distilled water and were dried on sterile filter paper for cuticle antisepsis as per the method described by Angelo et al. 2010 and Drummond et al. 1973 [9] . Commercially available deltamethrin was diluted in distilled water for preparation of five different working concentrations i.e., 25 ppm, 50 ppm, 70 ppm, 105 ppm and 200 ppm to perform AIT. The stock solution of the control group was prepared using distilled water. The ticks were immersed in 10 ml of concentrations of deltamethrin for 2 minutes with gentle agitation. The ticks in the control group were immersed in distilled water. The ticks were then dried on filter paper and transferred to Petri dishes having Whatman filter paper number no. 1. All the Petri dishes with treated ticks were maintained at room temperature for 24 hours and then each single female tick was transferred to a sterile glass test tube, covered with muslin cloth and kept in a BOD incubator at 28 0 C and 85±5% RH. (FAO, 2004; Gonçalves, et al., 2007) [10, 16] . The ticks were observed daily for the reproductive index by oviposition and mortality up to 15 days by observing their pedal reflexes. The reproductive index and percent inhibition of oviposition were calculated using the formulas (FAO 2004, Gonçalves et al., 2007) [10,16] .

Reproductive Index (RI) = egg mass weight/engorged tick weight Percentage inhibition of oviposition (%IO) = [(RI control-RI treated)/RI control x 100]

Results and Discussion

In the present study the resistance to the deltamethrin in R. microplus ticks from five talukas of Nagpur district of Maharashtra analyzed by AIT revealed the high level of resistance (100%) to deltamethrin at recommended concentration. The deltamethrin concentrations were increased up to 200 ppm still showing the resistance in R. microplus ticks. Even at 200 ppm concentration of deltamethrin no mortality of adult female ticks was observed.

After performing AIT, it was observed that Rhipicephalus microplus female ticks from all the five talukas of Nagpur district of Maharashtra state were found resistant against deltamethrin by considering the parameters viz; adult mortality, reproductive index, inhibition of oviposition and egg hatchability of female ticks. The data of AIT from Nagpur, Kamptee, Ramtek, Kalmeshwar and Umred is enumerated in Table 1. The dose-dependent response of deltamethrin in engorged Rhipicephalus microplus female ticks from all five talukas of Nagpur district of Maharashtra showed the mean percent mortality of engorged female ticks against 25, 50, 70, 105 and 200 ppm of deltamethrin was 0.00 in all the concentrations. The reproductive index (RI) of treated female ticks from all five talukas was reduced at increased concentrations of deltamethrin however RI in the control group was slightly higher as compared to treated female ticks with no more significant difference from respective taluka whereas the calculated values of percent inhibition of oviposition of treated female ticks was increased at higher concentrations of deltamethrin. The percent inhibition of oviposition in all the groups of treated female ticks were non-significant in all the concentrations of deltamethrin. The hatchability of eggs of treated female ticks was observed visually by counting a number of eggs in the treatment group and the hatching of larvae in control group showd no significant difference from all five talukas of Nagpur district of Maharashtra. In the present study, the resistance status of deltamethrin in Nagpur district of Maharashtra was observed up to 200 ppm concentrations even though the recommended concentration of deltamethrin is 25ppm. The ticks found 100 percent resistant to 25 ppm, 50 ppm, 70 ppm, 105 ppm and 200 ppm concentration of deltamethrin which is a big threat for tick control programme. To notice the mortality of ticks up to LC 50 and LC 95 the concentrations of deltamethrin have to be increased up to a very high level. The increased deltamethrin concentrations in my opinion are against the environmental safety hence the present study was restricted only up to 200 ppm. (Table 1.).

Discussion

The presence of deltamethrin resistance was reported in R. microplus ticks from many countries including India. High level of resistance against deltamethrin in R. microplus ticks was reported by, Singh et al. (2010) [36] from Punjab and Sharma et al. (2012) [32] from six agro-climatic regions of India. An elevated level of resistance against deltamethrin in R. microplus was reported worldwide as well as in India however the low level of resistance against deltamethrin in R. microplus was reported by Jyothimol et al. (2014) [20] from Kerala, Gaur et al. (2016) [13] and Gupta et al. (2023) [19] from Haryana and Rajasthan (Tohana tick isolates), Chigure et al. (2018) [7] from Uttar Pradesh, Krishnamurthy et al. (2019) [22] from Karnataka. Dadas (2015) [8] . Bharkad (2019) [5] and Khating (2023) [21] reported a higher level of resistance against deltamethrin in R. microplus ticks from different regions of Maharashtra after performing different bioassays. Whereas, Gaur et al. (2016) [13] from Haryana and Rajasthan (Agorha isolates), Gupta et al. (2022) [18] from Himalayas of India also reported the susceptibility of R. microplus ticks against deltamethrin. In India Ahanger et al. (2015) [2] from Jammu and Kashmir reported level I-IV resistance with LC 50 at 25.4-3868.8 ppm. Level I-II resistance of deltamethrin with LC 50 was reported by Gosh et al. (2015) [15] from Bihar at 27.6-467.1ppm, Shyma et al. (2015) [33] from Gujrat at 60.05-241.9 ppm, Sagar et al. (2020) [31] from Madhya Pradesh at 45.7-1829 ppm, Fular et al. (2021) [11] from Uttarakhand at 122.0-390.4 ppm. . Similarly Gupta et al. (2022) [18] from Himalayas determined level II-III resistance at 12.6-679.5 ppm after performing AIT. The reports from other countries revealed the presence of resistance against deltamethrin in Rhipicephalus microplus ticks reported by by Rodriguez et al. (2006) [29] from Mexico, Chevillon et al. (2007) [6] from New Caledonia, Vudriko et al. (2016) [40] from Uganda, Bandara and Karunarathe. (2017) [4] from Sri Lanka, Valsoni et al. (2020) [37] from Brazil and Villar et al. (2020) [39] from Colombia by performing AIT. Amongst different tick control methods use of chemical acaricides is widely used, hence there is always the risk of developing resistance (Sagar et al. 2020) [31] . In synthetic pyrethroid preferably deltamethrin is mostly used by Indian people (Kumar et al. 2020). Repeated applications of deltamethrin for control of ticks has resulted in selection of resistant tick populations in almost all tropical and sub-tropical countries (Mendes et al., 2007; Rodríguez-Vivas et al., 2014) [26,30] .

Conclusion

In the present study, it was observed that the commonly used deltamethrin for tick control programme in Nagpur district of Maharashtra developed resistance in R. microplus ticks infesting cattle. Also the increased deltamethrin concentrations beyond the recommended levels may be hazardous to human and animal health, nature creatures and may be responsible for environmental pollution so the studies may be conducted to incorporate biological control agents as an alternative to synthetic pyrethroids. Extension education and awareness amongst the farmers should be implemented to avoid haphazard and indiscriminate use of deltamethrin to overcome the problem of acaricidal resistance.

Acknowledgement

AcknowledgmentsThe authors are thankful to Department of Veterinary Parasitology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur (M.S.) for providing facilities.

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